Department of Industrial Chemistryand Materials

Recupero di biofenoli dalle A.V. contecniche di cromatografia

Leonardo Setti

w o r k s h o pAcque di vegetazione olearie:

trattamento e valorizzazione nel rispettodell’ambiente

Ottobre 23, 2007 – ENEA Centro Ricerche Casaccia

Integrated valorization processIntegrated valorization process

RECOVERY CHEMICALS

BIO-TRASFORMATIONCHEMICALS

BIOFUELS

(Biogas, bioethanol, biodiesel,…)

COMPOSTING FERTILIZERS

WASTES

Step 1

Step 2

Step 3

anthocyanins

Cross-Cross-sectional picture sectional picture of a green or black olive drupeof a green or black olive drupe

Phenolic acidsPhenolic acidsfor structuralfor structuralpolymerspolymers

? ? Phytoalexin secoiridoids oleuropeinPhytoalexin secoiridoids oleuropein

Source: Soler-Rivas et al. (2000) J. Sci. Food Agric. 80, 1013

Olive oil

Olive oil productionOlive oil production

Olive oil millingwastes83%

17%

Husks and waste waters

4 x 106 ton/y in Italy

30 x 106 ton/y in theMediterranean area

Ready for theextraction

Not Ready for theextraction

BiophenolsBiophenols in the Olive in the Olive milling wastesmilling wastes

4Verbascoside

Soluble Biophenols 1290-8760 mg/kg pulp of fresh olives

Soluble-Esterified Biophenols 570-2320 mg/kg pulp of fresh olives

Insoluble-bound Biophenols 1070-1620 mg/kg pulp of fresh olives

Xil

GalA

XilX ilX il X ilX ilX il

GalA GalA GalA

OO

OH

H3CO

OH

OCH 3

OO

Ga l

OH

OH

OO

GalAGalA

Glu

A

D

DB

C

GalA GalA GalA GalA GalAXil

GalA

XilX ilX il X ilX ilX il

GalA GalA GalA

OO

OH

H3CO

OH

OCH 3

OO

Ga l

OH

OH

OO

GalAGalA

Glu

A

D

DB

C

G alA G a lA G a lA G a lA G a lA

Model of the Model of the primary cellprimary cell--wall wall of of dicots dicots (Carpita and (Carpita and GibeautGibeaut, 1993), 1993)

Model of the Model of the primary cellprimary cell--wall wall of of dicotsdicots

Source: Saulnier and Thibault (1999) J.Sci.Food Agric. 79, 396 Guillon et al. (1989) Carbohydr. Res. 190, 97

Galacturonic acid

arabinose

galactose

Ferulic acid

Rhamnose acid

Methyl-esterifiedgalacturonic acidAcetylatedgalacturonic acid

“smooth” region “hairy” region

Galactans

Arabinans

Diferulic acidbridge

Structure Structure of of sugar beet pectin sugar beet pectin ““hairyhairy”” regionregion

Source: Colquhoun et al. (1994) Carbohydr. Res. 263, 243

CellCell--wall polysaccharide structure hydrolysiswall polysaccharide structure hydrolysisof of dicotsdicots

A Arabinase

B Arabinase

C Glucosidase

arabinosidase

D Arylesterase

Hydrolytic enzymaticactivities

Ara Ara Ara Ara Ara

Ara Ara Ara Ara Ara Ara

Xil

GalA

XilX ilX il X ilX ilX il

GalA GalA GalA

OO

OH

H3CO

OH

OCH 3

OO

Ga l

OH

OH

OO

GalAGalA

Glu

A

D

DB

C

Ara Ara Ara Ara Ara

Ara Ara Ara Ara Ara Ara

Xil

GalA

XilX ilX il X ilX ilX il

GalA GalA GalA

OO

OH

H3CO

OH

OCH 3

OO

Ga l

OH

OH

OO

GalAGalA

Glu

A

D

DB

C

A ra A ra A ra A ra A ra

A ra A ra A ra A ra A ra A ra

MIXTURES OF VARIOUS ENZYMATIC ACTIVITIES MIXTURES OF VARIOUS ENZYMATIC ACTIVITIESFROM FROM AspergillusAspergillus niger niger and and TrichodermaTrichoderma longhibrachiatumlonghibrachiatum

cellulasecellulase

pectinasepectinase

0.02 0.02 –– 0.1 0.1 µµmolmol/ml*/ml*minmin

acqua+SANSA

0,0

5,0

10,0

15,0

20,0

0 1 2 3

Time (gg.)

Red

ucin

g S

ugar

(m

g/m

l)

Y

X

Z

CONTROL

HUSKS/WATER: 33% w/v

ENZYME CONC.: 1% v/v

pH: 4.88

Husks hydrolysisHusks hydrolysis

Husks + distilled water

The efficiency of the enzymatic hydrolysis was tested withthe husks of olive milling wastes

HPLC/DAD HPLC/DAD analysis analysis on OMW on OMW before before andandafter the treatment after the treatment with with Y Y enzymaticenzymaticsolutionsolution

SEBPs HTy

IBBPs Phenolic acidsFerulic, caffeic, syringic,coumaric

+48% +98% +118% -17% +100%

g/l

SEBPs HTy

glucosidase

esterase

D

E

OleuropeinGlucose

Elenolic acid

Hydroxytyrosol

The extraction of these promising compounds hasThe extraction of these promising compounds hasstill some prominent problems to solve at anstill some prominent problems to solve at anindustrial scale:industrial scale:

1. the distant location and the recovery of wastes on the territory;2. the low concentration of bio-phenols in comparison to the large

amount of wastes to be treated;3. the management of a large amount of wastes to be transported to

the extraction plant and the relative costs;4. the large amount of wastes to be treated in time due to the scarce stability of

the wastes and their seasonality;5. the high-costs for the special disposal of the exhausted waste after solvent

extraction.

FoodProcessingwastes

Extractioncompany

Wastedisposal

SHUTTLE PROJECTSHUTTLE PROJECT

Pifferi PG., Setti L., Bordado J.Plant for the treatment of waste watersEuropean Patent WO 03/004419 A1

European Project FAIR CT 97 3039European Project FAIR CT 97 3039““Natural antioxidants from olive millingNatural antioxidants from olive milling

processprocess””

OMWW

Enzymatic treatment

Solid wastes Step 1

Enzymatic

solution

Step 2

Adsorption Step 3 Liquid wastes

Desorption Step 4

Washing of the resin

Liquid wastes

Resin

Step 6

Regeneration

Step 5

Finishing

Concentrated

liquid

Dry powder

Centrifugation

Scheme Scheme of theof theadsorptionadsorption//desorption processdesorption process

OIL

LIQUIDWASTES

CONCENTRATEDANTIOXIDANT

EXTRACT

Solvent

Rigenerationsolvent

LIQUIDWASTES

CONCENTRATEDWASTES

OMWW

1 2 34

56

78

9

RESINS

Resins

Resins

Enzymaticsolution

SchemeScheme of the of the technological processtechnological process at a pilot at a pilotscale (1 mscale (1 m33/d) of olive /d) of olive milling waste watersmilling waste waters

Demonstrative plantat Tuyap Technologies (Istanbul, Turkey) (2002)

Adsorption step

Sorbent: strong anionic exchanger styrene-divynil benzene copolymer

Sorbent concentration: 6% w/vStirred tank reactor at 25°C

Desorption stepDesorption step

Sorbent concentration: 6% w/vSolvent: Ethanol 96% : HCl conc. (99:1)Stirred tank reactor at reflux of the solvent (~ 80°C)

Storage conditions for 68 d

Regeneration cycles Regeneration cycles of of adsorptionadsorption//desorptiondesorptionprocess usingprocess using the the same resin same resin stockstock

Yield of the pilot scale processYield of the pilot scale process

Recovery of Soluble Biophenols from OMWAverage adsorption rate: 54% +- 13% in 10 h timeAverage desorption rate: 78% +- 22% in 1 h timeAverage recovery: 41% +- 15%

Mass balanceFrom1 litre of OMW => 1.6 g of biophenols

0.8 g of Hydroxytyrosol

0.1 – 0.2 l of extract containing 8 – 16 g/l of biophenols and4-8 g/l of hydroxytyrosol

OMWW25 m3/d

Enzymaticsolution

12.5 kg/d

OLIVE OIL180 – 450 kg/d

CONCENTRATEDWASTES

? kg/dResins

1500 - 3000kg/d

Solvent

Ethanol 49500 liters/dChloridric acid 500 liters/d

Biophenols115.5 kg/d

LIQUIDWASTES<25 m3/d

Ethanolicextract

50000 liters/d

CONCENTRATEDEXTRACT

2500 – 5000 liters/d

Biophenolsconcentration 6 -12 g/l

Biophenols amount63.2 kg/d

Ethanol45000 liters/d

Regeneration SolventChloridric acid liters/d

Water

LIQUIDWASTES<25 m3/d

1 2 3 4,5 6

78

9

SchemeScheme of the of the dimensioned technologicaldimensioned technologicalprocessprocess at at an industrialan industrial scale scale

VITAFOODS - 5th VITAFOODS - 5th International Exhibition International Exhibition & & Conference Conference on on NutraceuticalsNutraceuticals and and Functional Foods Functional Foods - - Geneve Geneve 20022002

““In vivoIn vivo”” ACTIVITIES OF OLIVE OIL WASTE WATER EXTRACTS ACTIVITIES OF OLIVE OIL WASTE WATER EXTRACTS RICH IN HYDROXYTYROSOL (HT)RICH IN HYDROXYTYROSOL (HT)

DeptDept. Of . Of Pharmacological SciencesPharmacological Sciences, , UnivUniv. Of . Of MilanMilanProf. Claudio GalliProf. Claudio Galli

•HT is bioavailable in animals and humans: it is dose-dependently adsorbed after oral ingestion

IN ANIMALS:

General antioxidant: increases the plasma antioxidant potential

Lipid antioxidant: reduces “in vivo” biomarkers of lipidperoxidation (urinary excretion of isoprostanes, non enzymaticoxidation products of arachidonic acid)

IN HUMANS:

Lipid antioxidant: increases urinary isoprostanes

Antithrombotic: reduces thromboxane formation

ConclusionsConclusions

1. Soluble biophenols can be extracted inquantitative way from agro-food wastes

2. Soluble biophenols were demonstrated to have animportant antioxidant powerful in vivo

3. Olive milling waste waters, winemaking wastesand mealmaking wastes could constitute a furtherindustrial resource of specific biophenols asnutraceutical compounds

4. The recovery/removal of the biophenols from thewastes could decrease the toxicity of the wastesthus improving any subsequent biological aerobicor anaerobic treatment